1. Field of the Invention
The present invention generally concerns procedures for generating transgenic flies for expression and study of a gene of interest. The invention particularly concerns processes for generating and using stocks of transgenic Drosophila that carry a mutant allele of a gene of unknown function related to the production of a new dominant phenotype, as well as methods for utilizing transgenic Drosophila to generate and study other genes, such as those encoding dominant-negative bacterial toxins.
2. Description of Related Art
Pharmacological research is hampered by the labor-intensive and extremely lengthy identification and systematic characterization procedures for new therapeutic compounds. A conventional process involves the screening of Thousands of individual compounds there are conventionally screened for a desired biological therapeutic benefit. Less than 1 in 10,000 of the synthetic compounds screened have ultimately been approved by the Food and Drug Administration. The cost approximates $200+ million per drug put into service. Natural products have provided the impetus for search of therapeutically effective pharmacological compounds for many years. Complex mixtures derived from cells, or their metabolites, are screened for biological activity, and the specific molecule possessing the activity is purified using the biological activity as the means for identifying the component of the mixture possessing the desired activity.
An alternative methodology has been to screen previously synthesized individual compounds saved in “libraries” in drug companies or research institutions. More recently, peptide or oligonucleotide libraries have been developed which may be screened for a specific biological function.
Because many of the existing therapeutics have been identified accidentally, their mechanism of action is not well understood. A more effective approach to identifying new molecular compounds effective against various disease conditions requires precise knowledge of the molecular defect underlying a given disease, and of the cellular pathways and processes of action. This is a weakness inherent in the current screening methodologies of compound libraries employing cell-free and in vitro cell-based assays.
It should also be noted that high throughput screening does not actually identify a drug, but merely high-quality “hits” or “leads” that are active at a relatively low concentration. This powerful screening tool also suffers from a number of limitations like bioavailability, toxicity and specificity. Subsequent studies are required before such a compound metamorphosizes into a therapeutically useful drug.
Screening of compound libraries with cell-free and in vitro assay systems has intrinsic limitations and weaknesses. For example, in cell-free systems, screening is limited to single target molecules, and they do not provide an inherent test for either specificity of the interaction, or of the toxicity of the test compound. But, perhaps most importantly, cell-free systems do not identify intermediary targets in signaling pathways made up of a hodge-podge of cell membrane, cytoplasmic, and nuclear-based components not present in cell-free systems.
Cell-based assays have several advantages over cell-free systems, the first being the capacity of self-replication. Moreover, the interactions occur in a biological context hopefully more closely mimicking the normal physiological conditions in vivo. Bioavailability and cytotoxicity are more easily assessed, but they often provide inadequate similarity to the in vivo disease conditions developing in multicellular tissues.
With the current screens using cell-free or in vitro cell-based assays, these differentiation end-points are difficult to assess and cell number or cell mass may be the more appropriate assay for their high-throughput designs. This is due to the fact that current screening methodologies can not easily discriminate growth arrest due to differentiation from other antiproliferative or simple cytotoxic effects.
Whole embryo cultures have also been used to screen for chemical effects in, for example, rodents and chickens. Adverse embryonic outcomes (malformations or embryotoxicity) are directly related to the serum concentration of the compound being tested. These serum concentrations can be directly compared to the serum concentration in the human. Whole embryo culture systems are problematic in that they result in large numbers of in vivo false-positives, and development within the cultures is limited to the very early stages of embryogenesis.
Similarly, the nematode C. elegans is frequently utilized as a model organism for the genetic dissection of developmental controls and cell signaling. However, in C. elegans there are no genetically sensitized systems available that permit reliable detection of even a two-fold reduction in a signaling process caused by either a chemical compound or a mutation in a gene. Although C. elegans can be grown in microtiter plates, the phenotypic screens are markedly limited. Also, chemical compounds would necessarily be administered by feeding, and would thus possess all of the aforementioned inherent disadvantages.
Another widely-utilized model genetic system is yeast. Although yeast are easily maintained and can readily be grown in large numbers, they are a simple, single-celled organism and thus possess the inherent limitation of being incapable of replicating a complex, multi-cellular system. Although the yeast system offers a comparatively higher throughput, it possesses inherent limitations, as most disease conditions are dependent upon cell-cell interactions within tissues that cannot be modeled in yeast. Finally, and most importantly, the overall degree of conservation of signaling pathways between yeast and human is significantly lower than that between Drosophila and humans.
Studies in the fruit fly Drosophila melanogaster have altered our estimate of the evolutionary relationship between vertebrate and invertebrate organisms. Key molecular pathways required for the development of a complex animal, such as patterning of the primary body axes, organogenesis, wiring of a complex nervous system and control of cell proliferation have been highly conserved since the evolutionary divergence of flies and humans. When these pathways are disrupted in either vertebrates or invertebrates, similar defects are often observed. The utility of Drosophila as a model organism for the study of human genetic disease is now well documented. Developmental defects such as the mesenchymal malformations associated with Saethre-Chotzen syndrome (Howard et al. 1997), formation of intracellular inclusions in polyglutamine-tract repeat disorders such as spinocerebellar ataxia and Huntington disease (Fortini and Bonini 2000), and loss of cellular-growth control and malignancy resulting from mutations of tumor suppressor genes (Potter et al. 2000) have been analyzed effectively using Drosophila as the model genetic system. The many basic processes that are shared between Drosophila and humans, in conjunction with the recent completion of the Drosophila genomic sequence, provide the necessary ingredients for launching systematic analyses of human disease-causing genes in Drosophila. 
The value of Drosophila as a screening system for evaluating the biological activities of chemicals has been well documented (see e.g., Schulz, et al, 1955. Cancer Res. 3(suppl.): 86-100; Schuler, et al., 1982. Terat. Carcin. Mutag. 2:293-301). Small numbers of chemical substances are administered to larvae or flies by feeding, and flies are then analyzed for survival and for phenotypic alterations. Although these conventional tests show the potential use of Drosophila as a tool to analyze the function of small molecular weight compounds, these methods neither permit high-throughput screens, nor permit the directed search for small molecular weight compounds that interfere with a specific morphogenetic pathway related to a human disease condition. Application of compounds by feeding requires relatively large amounts of the substance, and its uptake by the larvae and thus its final concentration is, at best, difficult to control. Furthermore, application by feeding does not permit automation of the procedure necessary for high-throughput analysis (Ernst Hafen, United States Patent Application, 20020026648).
The Drosophila epidermal growth factor (EGF)-receptor tyrosinekinase (EGF-R) controls a large array of cell-fate choices throughout the life cycle (Schweitzer, R. & Shilo, B. Z. (1997), Perrimon, N. & Perkins, L. A. (1997)) and can be activated by multiple ligands (Moghal & Sternberg (1999)). Among them, Vein is directly secreted to signal to adjacent cells (Schnepp, et al (1996)), whereas other EGF ligands such as Spitz (Spi) (Rutledge, et al (1992)) and Gurken (Grk) (Neuman-Silberberg & Schupbach (1993)) are similar to the human transforming growth factor a and are initially expressed as membrane-bound precursors. Numerous studies have provided corroborating evidence that these latter precursor ligands are initially inert and depend on two accessory membrane proteins, Rhomboid (Rohr) and Star, to be processed into active diffusible forms (Schweitzer et al (1995); Guichard Et Al (1999); Pickup & Banerjee (1999); Guichard Et Al (1999); Bang & Kintner (2006); Guichard Et Al (2000); Lee Et Al (2001); Urban Et Al (2001); Golembo Et Al (1996)). Rho is a predicted seven-pass transmembrane protein (Bier et al (1990)), and Star is predicted to be a type II single-pass transmembrane protein predominantly localized in the endoplasmic reticulum (ER) (Pickup & Banerjee (1996); Kolodkin et al (1994)), which acts as an obligate partner of Rho to activate EGF-R signaling in a cell nonautonomous fashion (Bier et al (1990)). Recent studies show that Star is necessary for Spi to translocate from the ER to the Golgi apparatus, where it is directly cleaved by Rho, a novel type of intramembrane serine protease (Lee Et Al (2001); Urban Et Al (2001); Tsruya et al (2002)). Unlike the Egf-r, spitz (spi), and Star genes, which are expressed ubiquitously in most epidermal cells, rhomboid (rho) is expressed in a highly localized and dynamic pattern (Bier et al (1990)) that correlates with the in situ activation pattern of mitogen-activated protein kinase (MAPK), an essential downstream component of all tyrosine kinase receptors (Gabay et al (1997) Science 277; Gabay et al (1997) Development 124; Bier (1998)). This latter observation suggests that Rho provides the appropriate restricted spatial and temporal activation for membrane-bound EGF ligands. A good example of the localized activity of Rho is provided by the wing disc, in which the restricted expression of rho in longitudinal stripes controls the commitment of these cells to the vein fate through the activation of EGF-RMAPK signaling. Thus, rhove mutants, who fail to express rho in vein primordia, lack sections of veins, whereas ubiquitous ectopic expression of rho converts the entire wing blade into a single solid vein (Bang & Kintner (2000); Lindsley, D. & Zimm (1992); Sturtevant et al (1993)).
Current methods for generating gain-of-function mutations are of two general sorts.
1) Structure/function studies in which mutant forms of a gene-x are created in vitro by one of a several of existing methods for making site directed mutations and then introduced into an organism to assay the function of the mutated gene.
2) Systematic screens for mutant alleles of the endogenous gene-x using one of a variety of mutagens.
These two types of analysis are typically very labor intensive and can only recover rather limited numbers of mutations. For example, in the case of Inventors' structure/function analysis of the Drosophila sog gene, one person spent approximately two years generating a collection of 23 mutant forms of the gene, which were then transformed into flies to obtain several independent transgenic lines of flies carrying each construct. These mutant sog constructs were then misexpressed in the wing to test for the function of the mutated genes. Using this approach Inventors identified two activities of Sog which had not been previously known. Thus, whereas misexpression of wild-type sog during wing development causes a mild loss-of vein phenotype (FIG. 1C; Yu et al., 1996), misexpression of one mutant truncated form of sog—referred to as supersog—generates more severe wing patterning defects (FIG. 1D; Yu et al., 2000), while misexpression of a second truncated form induces production of ectopic wing veins (Yu et al., manuscript in preparation). Inventors and other investigators have also conducted several different systematic screens for mutations in the endogenous sog gene, which cumulatively amounted to at least one year of work by a single person. These tedious screens lead to the isolation of only null and partial loss-of-function sog alleles (Wieschaus et al., 1984; Ferguson et al., 1992; Francois et al., 1994).
What is needed is a method that could be employed to generate dominant alleles of a wide range of genes using various mutagens to provide insight to the function and mechanism of action of novel genes. This approach should be of particular utility in investigating the function of human disease genes, which have no known functional motifs but have homologues in Drosophila (Reiter et al (2001)). Furthermore, the method should be applicable to any organism in which it is possible to misexpress transgenic constructs at high levels in a conditional fashion.